HPA Presentation 2 white background

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INVESTIGATION OF CEREBROSPINAL FLUID BSOP 27
National Standard Methods CSF SOP 27 How well
did we do in practice? 2 case histories
examined Evaluations and Standards Laboratory
Centre for Infections Sheffield Teaching
Hospitals 20 May 2005
Valerie Bevan Trevor Winstanley Rob Townsend
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INVESTIGATION OF CEREBROSPINAL FLUID BSOP 27
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Evaluations and Standards LaboratoryOrganisation
Chart
April 2005
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Infrastructure to producing Standard
Methods(Valeries slide)

• Staff in Standards Unit
• Director 10
• Head of Standards Unit (80)
• Two staff write, edit documents, co-ordinate
  process
• SOP Co-ordinator (document control, formatting,
  reference manager)
• Information Officer (20 - issues passwords,
  places docs on website)
• Office Manager (10 - organizes meetings, sends
  out all documents for meetings)
• Working Groups
• Medical, clinical and biomedical scientists
• National AMM, CVN, ACM, IBMS, SGM, UK wide,
  Pathlinks, Leeds, other networks
• All day meetings every two months
• Virology Working group
• FWE group

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Standards Unit (Valeries slide)

• SOPs current position
• 109 clinical microbiology
• 37 food water and environmental
• 50 media
• 42 guidance notes

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Global access Over 1000 passwords issued for the
Extranet site Password requests from 48
countries outside of the UK
Access to National SOPs (Valeries slide) New
website! http//hpa-standardmethods.org.uk
Versions in Adobe PDF www.HPA.org.uk www.evalua
tions-standards.org.uk Versions in Microsoft
Word Password protected Drafts SOPs for review
6 Over to Trevor
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Investigation of Cerebrospinal FluidNational
Standard Method
1st case history

• Dr Trevor Winstanley
• Royal Hallamshire Hospital
• Sheffield UK

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Specimen (Trevors slide)

• Cerebrospinal fluid
• Tuberculosis and HIV infection endemic in Somalia
• Unknown HIV status
• Specimen and request form labelled Category 3
• increased risk of containing organisms that may
  cause severe human disease and present a serious
  hazard to laboratory workers

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Patient (Trevors slide)

• 1994
• Male, 9 y
• Somalian
• In England for 3 months
• Headaches, vomiting, fever, diplopia
• ? tuberculous meningitis

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Specimen (Trevors slide)

• Cerebrospinal fluid
• Tuberculosis and HIV infection endemic in Somalia
• Unknown HIV status
• Specimen and request form labelled Category 3
• increased risk of containing organisms that may
  cause severe human disease and present a serious
  hazard to laboratory workers

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Safety (Trevors slide)

• Risk assessment laboratory safety protocols
  followed
• Class 1 biological safety cabinet at CL3
• capture and retain airborne particles and protect
  laboratory worker

• Centrifuge with sealed buckets
• opened in Class 1 cabinet
• All media appropriately labelled

Over to Val
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Tuberculous meningitis (Valeries slide)

• CSF may be infiltrated with lymphocytic cells
• Insidious clinical manifestations
• Rare in the UK
• Considered in areas of high TB prevalence and
  in patients from high risk groups
• Examination for AAFB or mycobacterial genome if
  specific indication
• Rapid tests are useful if positive, but culture
  remains the gold standard for diagnosis
• HG3, CL3
• Think of other unusual organisms

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Examination of CSF for meningitis (Valeries
slide)

• Complete cell count
• Differential leucocyte count
• Examination of Gram-stained smear
• Culture for pathogens
• Determination of glucose and protein concentrat
  ions
• PCR where appropriate

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Normal CSF values (Valeries slide)
12 Over to Trevor
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Cell Count Results (Trevors slide)

• Total cell count 440 x 106 / L
• White cell count 400 x 106 / L (0-10)
• Differential 40 polymorphs
• 55 lymphocytes
• 5 undifferentiated
• Protein 0.1 g/L (0.2-0.4)
• Glucose 1.6 mmol/L ( 60 plasma)

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Results (Trevors slide)

• Ziehl Neelsen film negative
• Mycobacterium culture negative
• India ink stain negative
• Cryptococcal Ag test negative
• Sabouraud culture negative
• Gram film No organisms seen

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Culture (centrifuged deposit) (Trevors slide)

• Bacteria
• Columbia BA (5) ANO2 _at_ 37oC
• Heated CBA AER CO2 (7) _at_ 37oC
• Hartleys digest broth 0.1 glucose _at_ 37oC
• Cryptococcus neoformans
• Sabouraud agar AER _at_ 30oC
• Mycobacterium spp.
• Löwenstein-Jensen Kirschners

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Further tests (Trevors slide)

• Aliquot mixed with equal volume of buffered
  formalin (4) and left for 10 min
• CSF cell counts
• Protein and glucose (Clinical Chemistry)
• Cryptococcal Ag test

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Culture media, conditions and organisms
(Valeries slide) For all specimens
Broth cultures are not recommended as a
significant positive yield is rarely achieved and
contamination is frequent
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Broth Cultures? (Valeries slide)

• Broth cultures are not recommended
• Significant positive yield is rarely achieved and
  contamination is frequent (Dunbar et al, 1998)

Over to Trevor
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Bacterial culture results (Trevors slide)

• CL3 accommodation
• Columbia BA ANO2 negative after 48 hours
• Chocolate CBA CO2 negative after 6 days
• Hartleys Digest Broth clear at 6 days
• Sub-cultured in Class 1 safety cabinet
• Columbia BA ANO2
• Chocolate CBA CO2

Over to Val
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Broth sub-cultures (Trevors slide)

• No growth _at_ 24 h
• 1 mm diameter colonies _at_ 48 h aerobic

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Gram film (Trevors slide)

• Small (0.5 µ) Gram negative cocci
• Oxidase positive ? Neisseria sp.
• N.meningitidis is in Hazard group 2
• At this time, local policy did not dictate the
  use of full
  containment level 3 conditions
  

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Susceptibility tests (Trevors slide)

• No growth on Lysed Blood Agar after 24 h
• Identity as Neisseria sp. questioned
• Cocco-bacillary forms seen on Gram film

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Biochemistry (Trevors slide)

• Rapid urease test positive
• Provisionally identified as Brucella sp.

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Biochemistry (Trevors slide)

• We did not attempt further identification
• Others have misidentied Brucella sp. as Moraxella
  phenylpyruvica
• Confirmed as B.melitensis by Portsmouth PHL

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Since broth sub-culture (Trevors slide)

• Cultures processed on open bench
• Provisional identification of Brucella sp. caused
  alarm
• an understatement
• infectious aerosol 10-100 organisms
• 7 laboratory workers handled cultures
• A further 4 handled specimens

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What did we do? (Trevors slide)

• Took base-line sera
• Post-exposure prophylaxis of doxycycline
  rifampicin (3 weeks)

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Consequences (Trevors slide)

• Pharmacy ran out of rifampicin
• The mens urinal turned red!
• Luckily, nobody was infected
• We learned some lessons

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Brucella sp. (Trevors slide)

• Zoonosis
• B.melitensis (goats and cattle) B.suis (swine)
  B.abortus (cattle) B.canis (dogs)
• Mediterranean Europe and Africa, Middle East,
  India, Central Asia, Mexico, Central and South
  America

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Transmission (Trevors slide)

• Ingestion, inhalation
• Direct contact
• cuts and abrasions, mucous membranes
• Laboratory exposure
• aerosolisation
• Bioterrorism
• high morbidity, protracted illness

Over to Val and Rob
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INVESTIGATION OF CEREBROSPINAL FLUID BSOP 27
Over to Rob
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A Clear Case ofMeningitis?
2nd case history

• Dr Rob Townsend MBCHB MSc DTMH
• Microbiology SpR/Lecturer
• Sheffield Teaching Hospitals

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The Patient (Robs slide)

• Mrs JM 30 Yr Old Admitted Royal Hallamshire
  Hospital, Sheffield, 2001
• Approx 24 hours History of
• Headache
• Neck Stiffness
• Photophobia

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History (Robs slide)

• No Rash noted
• Denies any prior symptoms
• No dysuria
• No sore throat
• Came on over hours the previous day
• Other family members well
• No pets
• No recent holiday

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Examination (Robs slide)

• Patient was noted to be Pyrexial
• Looked unwell but GCS 15/15
• Examination
• Neck stiffness clinically
• No rash
• No Papilloedema
• Remainder of examination unremarkable

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Investigations (Robs slide)

• Raised WCC on full blood count
• Blood cultures sent
• Lumbar Puncture performed
• Which Looked Like.

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Gin Clear ! (Robs slide)
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Lumbar Puncture (Robs slide)

• Cell count
• Total cell count lt1/hpf
• White cell count lt1/hpf
• Protein 0.5g/l
• Any Thoughts?

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Treatment (Robs slide)

• Given Cefotaxime in AE prior to ward admission
• Cefotaxime continued BUT Given CSF result
• ? Stop as it may be viral?
• ? Stop as it may not be meningitis?
• Continued Cefotaxime over night to review next day

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Next Day (Robs slide)

• Blood cultures CSF no growth
• But Post Antibiotic
• Reviewed by Consultant
• Clinically meningitis
• Raised CRP
• History revisited
• Any thoughts?

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Added History (Robs slide)

• No recent holiday BUT only been in UK 6/12
• Previously from Zimbabwe
• History of recurrent infections inc
• Respiratory infections
• Candidiasis
• Telephoned micro. Please could you do a Gram
  Film?
• Not in our protocol but as you asked.

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(Robs slide)
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Conclusions (Robs slide)

• Pneumococcal meningitis on gram film
• Cultures No growth (prior antibiotics)
• Requires 14 days treatment
• Later tests
• HIV ve
• CD4 count lt100
• Importance of Gram films in immunesuppressed
  patients with no cells they may have no cells!
• Completed 14 days and made good recovery
• 1 month later started Anti-retrovirals

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How did standard methods fair?(Valeries slide)

• Brucella covered in SOP
• Broth Cultures?
• Broth cultures are not recommended
• Significant positive yield is rarely achieved and
  contamination is frequent (Dunbar et al, 1998)
• Gram stain? (refer to BSOP SP 8)
• CSF SOP states do Gram-staining method on ALL
  specimens except
• clotted specimens (see below)
• routine neurological specimens unless leucocyte
  counts are raised

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About Psychrobacter phenylpyruvicus(Valeries
slide)

• Rods, often coccobacilli. Usually occur in
  planes with one plane of division. Microscopy
  can differentiate Brucella species (very small
  coccobacilli) from P. phenylpyruvicus
• Brucella species can be misidentified as P.
  phenylpyruvicus in some commercial identification
  kits

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Where do we go from here with National SOPs?
(Valeries slide)

• Future work
• Front end clinical algorithms for bacteriology
  AND virology
• Standards for reporting?
• Reconsider format?
• What should we emphasise?
• How do we evaluate methods?
• How do we encourage people to comment?

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(Valeries slide)

• Your help is still needed!

We have a new website to make things easier for
you
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• www.hpa.org.uk
• www.evaluations-standards.org.uk
• http//hpa-standardmethods.org.uk
• valerie.bevan_at_hpa.org.uk

Acknowledgements to all in Evaluations and
Standards Laboratory Laboratory staff at
Hallamshire Patients